Distinct and targetable role of calcium-sensing receptor in leukaemia

Haematopoietic stem cells (HSC) reside in the bone marrow microenvironment (BMM), where they respond to extracellular calcium [eCa2+] via the G-protein coupled calcium-sensing receptor (CaSR). Here we show that a calcium gradient exists in this BMM, and that [eCa2+] and response to [eCa2+] differ between leukaemias. CaSR influences the location of MLL-AF9+ acute myeloid leukaemia (AML) cells within this niche and differentially impacts MLL-AF9+ AML versus BCR-ABL1+ leukaemias. Deficiency of CaSR reduces AML leukaemic stem cells (LSC) 6.5-fold. CaSR interacts with filamin A, a crosslinker of actin filaments, affects stemness-associated factors and modulates pERK, β-catenin and c-MYC signaling and intracellular levels of [Ca2+] in MLL-AF9+ AML cells. Combination treatment of cytarabine plus CaSR-inhibition in various models may be superior to cytarabine alone. Our studies suggest CaSR to be a differential and targetable factor in leukaemia progression influencing self-renewal of AML LSC via [eCa2+] cues from the BMM.

Both sexes were used. We did not notice any sex-dependent differences.
The patients were between the ages of 57-76.

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The use of BM samples from male and female patients with AML for xenotransplantation experiments was approved by the medical ethics committee II of the Medical Faculty of the University of Heidelberg (Approval number 2013 509N MA). The supernatants of bone marrow aspirates from patients with different haematological malignancies were provided by the biobank of the University Center for Tumoral Diseases in Frankfurt am Main. These experiments were approved by the Ethics Committee of the University Clinic of the Goethe University Frankfurt (Approval number 274/18 and SHN 5 2020). Informed consent was obtained.
For experimental design, we perform power analysis, typically specifying P = 0.05, two-tailed testing, and power (= 1-) of 80% (ie, given a particular difference in groups, we want an 80% chance of getting statistically significant results), using commercially available software packages (Statistical Solutions nQuery Advisor; http://www.statsol.ie/nquery/nquery.htm). Although these conditions vary, our control cohorts are usually designed to have fairly tight incidence curves, allowing us to be adequately powered with experimental recipient cohorts of average size n =10.
No data were excluded from the analysis.
All in vitro experiments were performed using at least 3 biological replicates. All experiments have been repeated independently with similar results. All other experiments were repeated 3-5 times. All attempts at replication were successful.
In mouse experiments, animals were randomly assigned to experimental groups. For in vitro studies, samples were randomly allocated to experimental groups.
The investigators were blinded to group allocation during data collection and analysis. Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.
After purchase from DSMZ or ATCC the cells were not reauthenticated.
Cells were regularly tested for mycoplasma contamination and were only used for experiments, if free of mycoplasma contamination.
No commonly misidentified lines were used.
The study did not involve wild animals.
Animals of different sexes were randomly assigned to experimental groups, while ensuring that an even distribution of gender was found amongst all cohorts.
The study did not involve field-collected samples.
All animal studies were performed in compliance with the guiding principles of the 'Guide for the Care and Use of Laboratory Animals' and approved by the local government (Regierungspräsidium Darmstadt, Germany), protocol number Cells cultured in suspension were harvested and centrifuged at 1200rpm for 5 minutes and washed with cell staining buffer to remove residual growth factors that may be present in the culture medium.
Adherent cell lines were treated with trypsin and further incubated in cell culture medium. Cells were centrifuged at 1200rpm for 5 minutes and washed with cell staining buffer by centrifugation.
The pellet of either suspension or adherent cells was resuspended in cell staining buffer and the concentration adjusted to 1 x 106 cells/mL in cell staining buffer.